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Jun 10, 2014 - (14-16) Acute myeloid leukemia (AML) represents the clonal expansion of hematopoietic precursors blocked at different stages of differe...
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A Small-Molecule Targeting the MicroRNA Binding Domain of Argonaute 2 improves the Retinoic Acid Differentiation Response of the Acute Promyelocytic Leukemia Cell Line NB4 Silvia Masciarelli,†,⊥ Roberto Quaranta,†,⊥ Ilaria Iosue,†,⊥ Gianni Colotti,‡ Fabrizio Padula,† Greta Varchi,§ Francesco Fazi,*,† and Alberto Del Rio*,§,∥ †

Department of Anatomical, Histological, Forensic & Orthopaedic Sciences, Section of Histology & Medical Embryology and CNR-National Research Council of Italy, Institute of Molecular Biology and Pathology c/o Department of Biochemical Sciences ‘‘A. Rossi Fanelli’’, Sapienza University of Rome, 00161 Rome, Italy § CNR-National Research Council of Italy, Institute for Organic Chemistry and Photoreactivity, 40129 Bologna, Italy ∥ Department of Experimental, Diagnostic and Specialty Medicine (DIMES), University of Bologna, 40126 Bologna, Italy ‡

S Supporting Information *

ABSTRACT: Argonaute proteins are pivotal regulators of gene expression mediating miRNAs function. Modulating their activity would be extremely useful to elucidate the processes governing small-RNAs-guided gene silencing. We report the identification of a chemical compound able to compete with Argonaute 2 miRNAs binding, and we demonstrate that this functional inhibition determines effects similar to Argonaute 2 shRNA-mediated down-regulation, favoring granulocytic differentiation of the acute promyelocytic leukemia cell line NB4 in response to retinoic acid.

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mRNA is achieved, but this is rare in mammals although common in plants.4 MiRNAs mainly recognize their target mRNAs in the 3′-UTR through limited base-pairing interaction between the miRNAs “seed” region (miRNA nucleotides 2 through 7 or 8 from the 5′-end) and the mRNA complementary sequences.4 Ago2 is essential shortly after birth6 and, as for other proteins of the family in different organisms from Schizosaccharomyces pombe to mammals, is emerging as involved in other processes beyond gene expression regulation via mRNA binding.2 For instance, Ago2 is found in the nucleus of mammalian cells where it can regulate transcription by RNA interference mechanisms7 or by interaction with chromatin modifiers,8 and it can even participate to DNA repair.9 Since each miRNA can target hundreds of mRNAs and it has been estimated that about 60% of the mRNAs possess one or more sequences predicted to interact with miRNAs,10 it is clear that the RNA-binding proteins (RBPs) mediating their action have an important effect on main cellular processes such as proliferation, differentiation, resistance to stress or drugs, and death, in normal and pathological conditions. Among the plethora of biological processes regulated by miRNAs is included hematopoiesis, which is a life-long, highly regulated, multistage process in which pluripotent self-renewing hema-

icroRNAs are 18−25 nucleotide-long non-coding RNAs, conserved throughout evolution, that play a central role in gene expression mainly by preventing translation of target messenger RNAs.1 Most miRNAs are transcribed as primary precursors (pri-miRNA, 1−3 kb long) by Polymerase II from intergenic or intragenic regions, then processed to produce the pre-mRNA (70−100 nt long) by the nuclear microprocessor complex, comprising Drosha and DGCR8, and exported to the cytosol by Exportin-5. Here an RNase III enzyme, Dicer, processes pre-miRNA hairpins into mature miRNAs in collaboration with TRBP.2 The functional strand of the resulting 22 nt-long duplex miRNA/miRNA*, named guide strand, is loaded onto the RNA-induced silencing complex (RISC) where TRBP is required for the recruitment of a member of the Argonaute (Ago) family of proteins and the formation of a ternary complex together with Dicer.3 Once incorporated into RISC, miRNAs guide the complex to the target mRNAs and repress gene expression by suppressing protein translation or by destabilizing the mRNA.4 The human genome encodes four closely related Ago proteins characterized by N (amino-terminal), PAZ (PIWI−Argonaute−Zwille), Mid (middle), and PIWI domains. The PAZ and the Mid domains anchor the miRNA, the N domain is involved in loading and unwinding of the duplex miRNA, and the PIWI domain is a RNAase H fold that, despite the remarkable homology among human Ago proteins, is able to perform endonucleolytic cleavage only in Ago2.5 Such cleavage occurs only when a perfect complementarity between the miRNA and the target © 2014 American Chemical Society

Received: November 22, 2013 Accepted: June 10, 2014 Published: June 10, 2014 1674

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topoietic stem cells (HSCs) give rise to all blood cell lineages. Differentiation of erythroid, granulocytic, monocytic, and megakaryocytic lineages is controlled by the coordinate action of transcription factors, chromatin modifying enzymes, and miRNAs that cooperatively regulate specific target genes expression.11−13 Thus, alteration of transcription factors and miRNAs levels as well as chromatin organization may affect proliferation, differentiation, and genetic stability of HSCs, resulting in myeloproliferative disorders and leukemia.14−16 Acute myeloid leukemia (AML) represents the clonal expansion of hematopoietic precursors blocked at different stages of differentiation. In acute promyelocytic leukemia (APL), PML-RARα fusion protein is able to recruit various epigenetic enzymes on retinoic acid (RA)-target gene promoters, causing their transcriptional silencing and differentiation block. Pharmacologic doses of RA dissociate this chromatin repressive complex from PML-RARα binding sites, thus restoring transcriptional activity and myeloid differentiation of APL blasts in vitro and in vivo.17,18 To date, APL represents a paradigm for the differentiation therapy of leukemia providing a rationale for the design of molecular targeting drugs. MiRNAs play a central role in myeloid cell differentiation through complex regulatory circuitries. Because of the importance of miRNAs in hematopoiesis19−21 and the fact that Ago2 plays a central role in the differentiation of erythroid and B lymphoid cell lineages,22 we investigated in a previous work the role of Ago2 in myeloid cell differentiation, using among other AML cell lines the APL model cell line NB4.23 We showed that Ago2 is down-regulated when NB4 cells are induced to differentiate to granulocytes by RA; accordingly, Ago2 down-regulation by expression of a shRNA driven by a lentiviral vector improved retinoic acid induced differentiation.23 This evidence further supports the central role that RBPs involved in miRNAs activity may play in physiological and pathological processes.24 There is an increasing interest in developing small molecules able to inhibit RISC loading,25 and Schmidt et al.26 reported mRNA-mimicking compounds that are able to bind the miRNA’s seed region within the active site of Ago2. On the same basis, but with a different molecular design approach, we envisioned a new strategy to inhibit the activity of Ago2 by means of cell-permeable small molecules able to functionally interfere with miRNA loading, as their use would allow circumventing problems of delivery and immunogenic response linked to the use of viral vectors. Consistently with the increasing availability of structural data on RBPs, 27 we used structure-based molecular design techniques by taking advantage of the recent crystallographic structure of the human full-length Ago2 in complex with miR20a28 (Figure 1a). By means of high-throughput docking screening, we selected a compound named BCI-137 because of its marked pharmacophore mimicking the 5′ end of miRNAs in the Ago2 Mid domain (Figure 1b,c and Supporting Information Figure 1). We analyzed by SPR assay whether BCI-137 efficiently binds to Ago2. The goodness of the linear regression in the Scatchard plot was assessed by calculation of the regression coefficient (r = 0.9733). We indeed found that the inhibitor binds to Ago2 with good efficiency (KD = 126 ± 11 μM; Figure 1d,e). We then verified that binding of BCI-137 to Ago2 would not interfere with protein stability in vivo by Western blot analysis. Indeed we observed the same amount of Ago2 protein in lysates from cultures treated or not with BCI137 and the same strong down-regulation upon RA-induced

Figure 1. Identification of a molecular compound interacting with the Mid domain of Ago2. (a) Model of the human Ago2 in complex with miR-20a based on the crystal structure (PDB ID: 4F3T).28 The N domain is shown in orange, the PAZ domain in green, the Mid domain in blue, the PIWI domain in maroon, linker regions in white, and the miR-20a in red. The black square identifies the region of the Mid domain used to set up the high-throughput molecular docking screening. (b) Chemical structure of the 2,3-dioxo-1,4-dihydroquinoxalin derivative BCI-137. (c) Molecular surfaces of the Mid domain binding site of Ago2 in complex with the miR-20a (left) and the representative binding mode of compound BCI-137 obtained with docking simulation (right). Compound BCI-137 forms a tight binding with several residues of the Mid domain, which is reminiscent of the uridine placement at the 5′ end of miR-20a (Supporting Information Figure 1). (d) Sensorgram showing the binding of BCI-137 to immobilized Ago2. The association phase (0−100 s) shows the injection of the following samples in HBS buffer (10 mM HEPES, pH 7.4; 150 mM NaCl; 0.005% surfactant P20): 0−16 s, 3.75 μM BCI137; 17−33 s, 7.5 μM BCI-137; 33−48 s, 15 μM BCI-137; 48−63 s, 30 μM BCI-137; 63−78 s, 60 μM BCI-137; 78−93 s, 120 μM BCI137; 94−100 s, 240 μM BCI-137. A single dissociation phase was measured by injecting HBS at a rate of 30 μL/min, starting at 100 s. (e) Scatchard plot of the interaction in panel d: for each concentration of the analyte, the RU and RU/c values were plotted and fitted by linear regression.

differentiation (Figure 2a). To determine experimentally if BCI137 was actually able to impair Ago2 loading of miRNAs, we performed RNA-immunoprecipitation (RIP) experiments using an antibody to immunoprecipitate Ago2 in lysates from proliferating or differentiating cells treated or not with BCI137 and then evaluating the amount of co-precipitated miRNAs (Figure 2b). At first, we analyzed the binding between Ago2 and miR-20a as in silico screenings were performed by using the crystallographic structure of Ago2 bound to this miRNA.28 As expected, since Ago2 itself is highly down-regulated in cells induced to differentiate by RA (Figure 2a), the amount of miR20a found in the immunoprecipitated material was reduced upon RA treatment. Importantly, the recruitment of miR-20a by Ago2 was also reduced in cells treated with the BCI-137 compound alone. We corroborated the results by assessing also miR-223, miR-26a, miR-107, and let-7a binding to Ago2, four 1675

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We have shown in our previous work that Ago2 regulates the expression of miR-155, a miRNA involved in myeloid differentiation, by localizing at the miR-155 host gene promoter and contributing to its heterochromatic silencing.23 We thus verified if BCI-137 was able to interfere with Ago2 activity on chromatin remodeling by testing miR-155 host gene promoter acetylation status in the presence or in the absence of BCI-137. Chromatin-immunorecipitation (ChIP) experiments demonstrated that treatment of NB4 cells with BCI-137 resulted in displacement of Ago2 from the miR-155 host gene promoter paralleling an increase in histone H4 acetylation in the same genomic region, (Figure 2c), further indicating that this molecule interferes with Ago2 activity, resulting in the same effects obtained by Ago2 shRNA-mediated silencing. Increasing doses of BCI-137 did not interfere with the proliferation or cell death rate of NB4 either in the presence or in the absence of RA (Figure 3a,b). However, we observed that in vitro treatment with the BCI-137 molecule was able to increase RA-dependent granulocytic differentiation, as evaluated by the analysis of the expression of the differentiationrelated miR-223 and miR-26a (Figure 3c), by morphological analysis (Figure 3d) and by NBT assay for the assessment of phagocytic and cytotoxic activity (Figure 3e). Indeed the combined treatment of RA plus BCI-137 compound compared to RA treatment alone resulted in higher induction of miR-223 and miR-26a, in augmented chromatin condensation and decreased cytosolic basophilia from a morphological point of view, as well as in an higher capacity of NBT reduction. In summary, by means of structure-based molecular design approach, we identified a small and cell-permeable molecule that is able to bind Ago2. Interestingly, we demonstrate that such compound has the biological effects expected on the basis of the prediction that it would functionally inhibit Ago2 since it produced effects similar to those observed by silencing Ago2 in the same cell line by lentiviral vector driven shRNA.23 Our work leads to two major conclusions. Improvement of APL cell differentiation obtained with the use of a combination of RA and BCI-137 could have relevance for the enhancement of RA efficacy response of non-APL AML subtypes. Even more significantly, the identification of small molecules that do not need to be delivered to the cell, with the ability to functionally inhibit Ago2, paves the way for studies in any kind of in vitro or in vivo system in which the role of Ago2 needs to be dissected and may constitute the basis of future therapeutic interventions for a wide set of pathologies.

Figure 2. Small molecule BCI-137 interferes with Ago2-nucleic acid interaction. (a) BCI-137 does not affect Ago2 protein levels. Ago2 protein expression was assessed by Western blot of total lysates of NB4 cells induced or not to differentiate by RA in the presence or in the absence of 100 nM BCI-137 for 48 h. Tubulin was used as loading control. (b) BCI-137 inhibits the binding of miR-20a, miR-223, and miR-26a to Ago2. Forty-eight hours after induction of differentiation, in the presence or in the absence of 100 nM BCI137, the cells were harvested and lysed, and Ago2 protein was immunoprecipitated by an anti-Ago2 antibody. The RNA in the immunoprecipitated material was extracted and analyzed by real-time PCR to evaluate the amount of the miRNAs co-precipitated with Ago2 (RIP). The histograms report the mean percentage decrease ± SEM, relative to the untreated sample, from two independent RIP experiments, calculated on the real time PCR values obtained by the ΔΔCt method using the RNU6B and the RIP inputs for normalization. (c) Upper panel: schematic representation of the genomic structure of human miR-155 gene. Arrows indicate the location of the primers used in the ChIP assay. Lower panel: histograms of the real time PCR performed to amplify miR-155HG regulatory region in ChIP assays carried out using an antiAgo2 antibody or an anti-acetyl-Histone-H4 antibody on chromatin samples prepared from NB4 cells treated or not with 10 μM BCI-137 compound. Error bars represent SD of two independent evaluations.

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METHODS

Molecular Modeling and Ligand Interaction Analysis. The crystal structure of human Argonaute-2 in complex with miR-20a (PDB ID: 4F3T) was used to perform molecular modeling, interaction analysis, and virtual screening experiments. The Protein Preparation Wizard routine of the Maestro Schrödinger software package (version 9.3) was used to prepare the protein downloaded from the Protein Data Bank. Gaps and missing loops in the protein structure were reconstructed with the Prime module (version 3.1). High-Throughput Molecular Docking Screenings. A 20 Å docking grid box was centered manually in the 5′-end region of the Ago2 Mid domain (Figure 1c). A high-throughput in silico screening with standard settings was performed by using the Asinex compound subset of the CoCoCo databases. A total of around 260,000 molecules were screened, visually inspected, and selected taking into account Ago2 Mid domain, drug- and lead-likeness, compound availability, chemical diversity, and synthetic accessibility. On the basis of these

miRNAs regulated during myeloid differentiation,20,29,30 which was also reduced by treatment with BCI-137 (Figure 2b and Supporting Information Figure 3). These results indicate that BCI-137 interferes with binding of miRNAs to Ago2. 1676

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Figure 3. Small molecule BCI-137 favors RA-induced differentiation of the APL cell line NB4. NB4 cells were induced to differentiate with 100 nM retinoic acid (RA) in the presence or in the absence of the indicated amount of BCI-137 (from 0.01 to 10 μM). (a) The proliferation curve and (b) prodium iodide staining show that BCI-137 (from 0.01 to 10 μM) does not interfere with cell proliferation or viability. (c) The differentiation-related miR-223 and miR-26a are expressed at higher levels in NB4 cells differentiated in the presence of 100 nM BCI-137. Real-time PCR analysis was performed by the ΔΔCt method using RNU6B as endogenous control for standardization; histograms represent the average ± SEM of three independent experiments. (d) Wright−Giemsa staining of NB4 cells indicates a more differentiated phenotype after 96 h of combined treatment with RA and BCI-137 (0.01 and 10 μM) compared to RA alone. (e) Cells treated as indicated for 96 h show a higher NBT reduction capacity. Histograms report the results of a representative experiment out of four. In the left panel are shown examples of light fields of the NBT assay where positive black cells are clearly distinguishable. criteria, nine molecules were selected and purchased for experimental testing. Chemical Identity, Sample Purity, and Spectral Data. BCI137, 2-(2,3-dioxo-1,2,3,4-tetrahydroquinoxaline-6-sulfonamido) propanoic acid, was purchased from Asinex Ltd. (compound ID BAS 07870068). Purity of the compound was ≥95%, as declared by the chemical vendor. 1H and 13C NMR spectra were recorded on a Varian spectrometer operating at 400 MHz for 1H and 100 MHz for 13C (Supporting Information, Figure 2 and methods). Surface Plasmon Resonance (SPR) Measurements. SPR experiments were carried out using a SensiQ Pioneer system (SensiQ, ICx Nomadics Inc.). The interaction of the immobilized protein with the analyte BCI-137 was detected by mass concentration-dependent

changes of the refractive index on the sensor chip surface. The changes in the observed SPR signal are expressed as resonance units (RU). Typically, a response change of 1000 RU corresponds to a change in the surface concentration on the sensor chip of about 1 ng of protein per mm2.31 See Supporting Information. Cell Cultures and Proliferation/Differentiation Assays. The NB4 cell line32 was purchased by DSMZ and maintained in RPMI 1640 medium supplemented with penicillin (50 U/mL)/streptomycin (50 μg/mL) solution, 2 mM L-glutamine, and 10% fetal bovine serum. See Supporting Information for experimental details. RNA Extraction and Real Time PCR. Total RNA from cells was extracted using a TRIZOL RNA isolation system (Invitrogen) according to the manufacturer’s instructions and reverse transcribed 1677

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with High Capacity RNA to cDNA Kit (Applied Biosystem). The quantification of Hsa-miR-26a and Hsa-miR-223 was carried out in ABI PRISM 7500 Sequence Detection System (Applied Biosystems) with TaqMan MicroRNA Assay (Applied Biosystems). ΔΔCt values were normalized with those obtained from the amplification of the endogenous U6 snRNA (Applied Biosystems). Western Blotting. Thirty micrograms of whole cell extract were separated by 4−15% Mini-Protean TGX precast gel (Bio-Rad) SDSPAGE and electroblotted to nitrocellulose membrane (Protran, Whatman). Immunoblots were incubated with antibodies to Ago2 (#2897, Cell Signaling); the anti-α-tubulin mouse monoclonal IgG (T5168 Sigma-Aldrich) was used to normalize the amount of the samples analyzed. The immunoreactivity was determined by the enhanced chemiluminescence (ECL) method (Amersham Biosciences). RNA-Binding Protein Immunoprecipitation (RIP) Assay. Total RNA from cells was immunoprecipitated using the Magna RIP kit (cat. no. 17-700, Millipore) following the manufacturer’s instructions with an anti-Ago2 antibody (Millipore, cat. no. 03-110) or control IgG. Binding of miRNAs to Ago2 was evaluated by qRTPCR; the quantification of Hsa-miR-20a, Hsa-miR-26a, Hsa-miR-223, Hsa-miR-107, and Hsa-let-7a was carried out in ABI PRISM 7500 Sequence Detection System (Applied Biosystems) with TaqMan MicroRNA Assay (Applied Biosystems). ΔΔCt values were normalized with those obtained from the amplification of the endogenous U6 snRNA (Applied Biosystems) and the respective input. See also Supporting Information. Chromatin Immunoprecipitation (ChIP) Assay. ChIP assays were performed as described by using antibodies anti-acetyl-HistoneH4 (H4Ac) (Millipore, cat. no. 06-866) and anti-Ago2 (Millipore, cat. no. 03-110).18,23 See Supporting Information.

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ASSOCIATED CONTENT

S Supporting Information *

Interaction diagrams of the 5′ end of miR-20a and BCI-137; Spectroscopic analysis of BCI-137; RIP quantification of let-7a and miR-107; Supplementary methods. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Author Contributions ⊥

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from the Italian Association for Cancer Research (AIRC): Emilia-Romagna AIRC Start-up grant 6266 (A.D.R.) and AIRC Start-up grant 4841 (F.F.).

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ABBREVIATIONS AML, acute myeloid leukemia; APL, acute promyelocytic leukemia; PML, promyelocytic leukemia; RA, retinoic acid; RARα, retinoic acid receptor α; AGO, argonaute; RBPs, RNAbinding proteins

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REFERENCES

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